Coherent X-ray diffraction imaging of chloroplasts from Cyanidioschyzon merolae by using X-ray free electron laser

Yuki Takayama, Yayoi Inui, Yuki Sekiguchi, Amane Kobayashi, Tomotaka Oroguchi, Masaki Yamamoto, Sachihiro Matsunaga, Masayoshi Nakasako

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Coherent X-ray diffraction imaging (CXDI) is a lens-less technique for visualizing the structures of non-crystalline particles with the dimensions of submicrometer to micrometer at a resolution of several tens of nanometers. We conducted cryogenic CXDI experiments at 66 K to visualize the internal structures of frozen-hydrated chloroplasts of Cyanidioschyzon merolae using X-ray free electron laser (XFEL) as a coherent X-ray source. Chloroplast dispersed specimen disks at a number density of 7/(10×10μm2) were flash-cooled with liquid ethane without staining, sectioning or chemical labeling. Chloroplasts are destroyed at atomic level immediately after the diffraction by XFEL pulses. Thus, diffraction patterns with a good signal-to-noise ratio from single chloroplasts were selected from many diffraction patterns collected through scanning specimen disks to provide fresh specimens into the irradiation area. The electron density maps of single chloroplasts projected along the direction of the incident X-ray beam were reconstructed by using the iterative phase-retrieval method and multivariate analyses. The electron density map at a resolution of 70 nm appeared as a C-shape. In addition, the fluorescence image of proteins stained with FlamingoTM dye also appeared as a C-shape as did the autofluorescence from Chl. The similar images suggest that the thylakoid membranes with an abundance of proteins distribute along the outer membranes of chloroplasts. To confirm the present results statistically, a number of projection structures must be accumulated through high-throughput data collection in the near future. Based on the results, we discuss the feasibility of XFEL-CXDI experiments in the structural analyses of cellular organelles.

Original languageEnglish
Pages (from-to)1272-1286
Number of pages15
JournalPlant and Cell Physiology
Volume56
Issue number7
DOIs
Publication statusPublished - 2015

Keywords

  • Chloroplast
  • Coherent X-ray diffraction imaging
  • Cyanidioschyzon merolae
  • Fluorescence optical microscopy

ASJC Scopus subject areas

  • Physiology
  • Plant Science
  • Cell Biology

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